The long-term objective of this project is to define the molecular mechanisms that regulate the traffic and functional activity of CFTR CL channels at the epithelial cell surface. This renewal application focus on the regulation of CFTR function by two members of the syntaxin family of membrane traffic regulators (i.e., syntaxins 1A and 3); each of which is expressed at the apical poles of colonic epithelial cells. Syntaxin 1A physically associates with CFTR CI channels and regulates CFTR CL currents in colonic ephithelial cells and heterologous expression systems. We propose that snytaxin 1A regulates CFTR function in one or both of the following ways: (ii) by regulating the numbers of CFTR molecules at the cell surface as part of the machinery that controls the intracellular traffic of CFTR and/or (ii) by directly modulating CFTR CI channels via protein-protein interactions. This proposal will be tested by pursuing 3 specific aims. First, we will verify that syntaxin 1A modulates CFTR CI current activity and determine if the regulation of CFTR CI currents by sntaxin 1 A correlates with changes in the numbers of CFTR molecules at the cell surface. We will also determine if syntaxin 1A directly regulates CFTR CI channels in excised membrane patches and planar lipid bilayers. The second specific aim is to define the structural basis for the physical interaction between syntaxin 1A and CFTR. We will map the relevant binding sites on each of these molecules and characterize the functional activities of syntaxin 1A mutants that lack the ability to bind CFTR. The regulation of this interaction by n-Sec 1 (i.e., a syntaxin 1A binding protein that I s also expressed in colonic epithelial cells) and by cAMP-dependent protein kinase (which phosphorylates both molecules) will be also be examined. the third aim is to define the specific roles that syntaxins 1A and 3 play in controlling apical membrane traffic (e.g., endocytosis and membrane recycling) in polarized colonic epithelial cells. Our results should provide novel insights into the molecular machinery that controls the traffic and functional activity of CFTR CI channels, which are defective or lacking in the most common genetic disorder among Caucasians (i.e, cystic fibrosis).